Abstract Identifying true DNA cellular barcodes among polymerase chain reaction and sequencing errors is challenging. Current tools are restricted in the diversity of barcode types supported or the analysis strategies implemented. As such, there is a need for more versatile and efficient tools for barcode extraction, as well as for tools to investigate which factors impact barcode detection and which filtering strategies to best apply. Here we introduce the package CellBarcode and its barcode simulation kit, CellBarcodeSim, that allows efficient and versatile barcode extraction and filtering for a range of barcode types from bulk or single-cell sequencing data using a variety of filtering strategies. Using the barcode simulation kit and biological data, we explore the technical and biological factors influencing barcode identification and provide a decision tree on how to optimize barcode identification for different barcode settings. We believe that CellBarcode and CellBarcodeSim have the capability to enhance the reproducibility and interpretation of barcode results across studies.